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ABSTRACT: Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP300 and CREBBP, are dynamically acetylated; (2) that nuclear acetyltransferases themselves are regulated, not on the protein abundance level, but by (de)acetylation; and (3) that the recently reported p53 co-activator and methyltransferase MLL3 is acetylated on five lysines during the DDR. For selected examples, protein immunoprecipitation and immunoblotting were used to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.
Cell cycle (Georgetown, Tex.) 05/2013; 12(11). · 5.36 Impact Factor
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ABSTRACT: Many cancers arise at sites of infection and inflammation. Cellular senescence, a permanent state of cell cycle arrest that provides a barrier against tumorigenesis, is accompanied by elevated proinflammatory cytokines such as IL1, IL6, IL8 and TNFα. Here we demonstrate that media conditioned by cells undergoing any of the three main forms of senescence, i.e. replicative, oncogene- and drug-induced, contain high levels of IL1, IL6, and TGFb capable of inducing reactive oxygen species (ROS)-mediated DNA damage response (DDR). Persistent cytokine signaling and activated DDR evoke senescence in normal bystander cells, accompanied by activation of the JAK/STAT, TGFβ/SMAD and IL1/NFκB signaling pathways. Whereas inhibition of IL6/STAT signaling had no effect on DDR induction in bystander cells, inhibition of either TGFβ/SMAD or IL1/NFκB pathway resulted in decreased ROS production and reduced DDR in bystander cells. Simultaneous inhibition of both TGFβ/SMAD and IL1/NFκB pathways completely suppressed DDR indicating that IL1 and TGFβ cooperate to induce and/or maintain bystander senescence. Furthermore, the observed IL1- and TGFβ-induced expression of NAPDH oxidase Nox4 indicates a mechanistic link between the senescence-associated secretory phenotype (SASP) and DNA damage signaling as a feature shared by development of all major forms of paracrine bystander senescence.
Aging 12/2012; · 5.13 Impact Factor
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Lenka Oplustilova,
Kamila Wolanin,
Martin Mistrik,
Gabriela Korinkova,
Dana Simkova,
Jan Bouchal,
Rene Lenobel,
Jirina Bartkova,
Alan Lau,
Mark J O'Connor,
Jiri Lukas, Jiri Bartek
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ABSTRACT: Impaired DNA damage response pathways may create vulnerabilities of cancer cells that can be exploited therapeutically. One such selective vulnerability is the sensitivity of BRCA1- or BRCA2-defective tumors (hence defective in DNA repair by homologous recombination, HR) to inhibitors of the poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response to PARP-1i. Here we addressed these issues using PARP-1i on 20 human cell lines from carcinomas of the breast, prostate, colon, pancreas and ovary. Aberrations of the Mre11-Rad50-Nbs1 (MRN) complex sensitized cancer cells to PARP-1i, while p53 status was less predictive, even in response to PARP-1i combinations with camptothecin or ionizing radiation. Furthermore, monitoring PARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confirmed the role of the multidrug resistance efflux transporters and its reversibility. More importantly, we demonstrated that shRNA lentivirus-mediated depletion of 53BP1 in human BRCA1-mutant breast cancer cells increased their resistance to PARP-1i. Given the preferential loss of 53BP1 in BRCA-defective and triple-negative breast carcinomas, our findings warrant assessment of 53BP1 among candidate predictive biomarkers of response to PARPi. Overall, this study helps characterize genetic and functional determinants of cellular responses to PARP-1i and contributes to the search for biomarkers to exploit PARP inhibitors in cancer therapy.
Cell cycle (Georgetown, Tex.) 09/2012; 11(20):3837-50. · 5.36 Impact Factor
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Thorkell Gudjonsson,
Matthias Altmeyer,
Velibor Savic,
Luis Toledo,
Christoffel Dinant,
Merete Grøfte,
Jirina Bartkova,
Maria Poulsen,
Yasuyoshi Oka,
Simon Bekker-Jensen,
Niels Mailand,
Beate Neumann,
Jean-Karim Heriche,
Robert Shearer,
Darren Saunders, Jiri Bartek,
Jiri Lukas,
Claudia Lukas
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ABSTRACT: Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.
Cell 08/2012; 150(4):697-709. · 32.40 Impact Factor
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Mads Daugaard,
Annika Baude,
Kasper Fugger,
Lou Klitgaard Povlsen,
Halfdan Beck,
Claus Storgaard Sørensen,
Nikolaj H T Petersen,
Poul H B Sorensen,
Claudia Lukas, Jiri Bartek,
Jiri Lukas,
Mikkel Rohde,
Marja Jäättelä
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ABSTRACT: Lens epithelium-derived growth factor p75 splice variant (LEDGF) is a chromatin-binding protein known for its antiapoptotic activity and ability to direct human immunodeficiency virus into active transcription units. Here we show that LEDGF promotes the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. Depletion of LEDGF impairs the recruitment of C-terminal binding protein interacting protein (CtIP) to DNA DSBs and the subsequent CtIP-dependent DNA-end resection. LEDGF is constitutively associated with chromatin through its Pro-Trp-Trp-Pro (PWWP) domain that binds preferentially to epigenetic methyl-lysine histone markers characteristic of active transcription units. LEDGF binds CtIP in a DNA damage-dependent manner, thereby enhancing its tethering to the active chromatin and facilitating its access to DNA DSBs. These data highlight the role of PWWP-domain proteins in DNA repair and provide a molecular explanation for the antiapoptotic and cancer cell survival-activities of LEDGF.
Nature Structural & Molecular Biology 07/2012; 19(8):803-10. · 12.71 Impact Factor
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ABSTRACT: Tumor suppressor PML is induced under viral and genotoxic stresses by interferons and JAK-STAT signaling. However, the mechanism responsible for its cell type-specific regulation under non-stimulated conditions is poorly understood. To analyze the variation of PML expression, we utilized three human cell types, BJ fibroblasts and HeLa and U2OS cell lines, each with a distinct PML expression pattern. Analysis of JAK-STAT signaling in the three cell lines revealed differences in levels of activated STAT3 but not STAT1 correlating with PML mRNA and protein levels. RNAi-mediated knockdown of STAT3 decreased PML expression; both STAT3 level/activity and PML expression relied on IL6 secreted into culture media. We mapped the IL6-responsive sequence to an ISRE(-595/-628) element of the PML promoter. The PI3K/Akt/NFκB branch of IL6 signaling showed also cell-type dependence, being highest in BJ, intermediate in HeLa, and lowest in U2OS cells and correlated with IL6 secretion. RNAi-mediated knockdown of NEMO (NF-κ-B essential modulator), a key component of NFκB activation, suppressed NFκB targets LMP2 and IRF1 together with STAT3 and PML. Combined knockdown of STAT3 and NEMO did not further promote PML suppression, and it can be bypassed by exogenous IL6, indicating the NF-κB pathway acts upstream of JAK-STAT3 through induction of IL6. Our results indicate that the cell type-specific activity of IL6 signaling pathways governs PML expression under unperturbed growth conditions. As IL6 is induced in response to various viral and genotoxic stresses, this cytokine may regulate autocrine/paracrine induction of PML under these pathophysiological states as part of tissue adaptation to local stress.
Journal of Biological Chemistry 06/2012; 287(32):26702-14. · 4.77 Impact Factor
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ABSTRACT: The conserved MRE11–RAD50–NBS1 (MRN) complex is an important sensor of DNA double-strand breaks (DSBs) and facilitates DNA repair by homologous recombination (HR) and end joining. Here, we identify NBS1 as a target of cyclin-dependent kinase (CDK) phosphorylation. We show that NBS1 serine 432 phosphorylation occurs in the S, G2 and M phases of the cell cycle and requires CDK activity. This modification stimulates MRN-dependent conversion of DSBs into structures that are substrates for repair by HR. Impairment of NBS1 phosphorylation not only negatively affects DSB repair by HR, but also prevents resumption of DNA replication after replication-fork stalling. Thus, CDK-mediated NBS1 phosphorylation defines a molecular switch that controls the choice of repair mode for DSBs.
EMBO Reports 05/2012; 13(6):561-8. · 7.36 Impact Factor
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ABSTRACT: The cellular DNA damage response (DDR) machinery that maintains genomic integrity and prevents severe pathologies, including cancer, is orchestrated by signaling through protein modifications. Protein ubiquitylation regulates repair of DNA double-strand breaks (DSBs), toxic lesions caused by various metabolic as well as environmental insults such as ionizing radiation (IR). Whereas several components of the DSB-evoked ubiquitylation cascade have been identified, including RNF168 and BRCA1 ubiquitin ligases, whose genetic defects predispose to a syndrome mimicking ataxia-telangiectasia and cancer, respectively, the identity of the apical E1 enzyme involved in DDR has not been established. Here, we identify ubiquitin-activating enzyme UBA1 as the E1 enzyme required for responses to IR and replication stress in human cells. We show that siRNA-mediated knockdown of UBA1, but not of another UBA family member UBA6, impaired formation of both ubiquitin conjugates at the sites of DNA damage and IR-induced foci (IRIF) by the downstream components of the DSB response pathway, 53BP1 and BRCA1. Furthermore, chemical inhibition of UBA1 prevented IRIF formation and severely impaired DSB repair and formation of 53BP1 bodies in G 1, a marker of response to replication stress. In contrast, the upstream steps of DSB response, such as phosphorylation of histone H2AX and recruitment of MDC1, remained unaffected by UBA1 depletion. Overall, our data establish UBA1 as the apical enzyme critical for ubiquitylation-dependent signaling of both DSBs and replication stress in human cells, with implications for maintenance of genomic integrity, disease pathogenesis and cancer treatment.
Cell cycle (Georgetown, Tex.) 04/2012; 11(8):1573-82. · 5.36 Impact Factor
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ABSTRACT: Nucleotide excision repair (NER) is an important DNA repair mechanism required for cellular resistance against UV light and toxic chemicals such as those found in tobacco smoke. In living cells, NER efficiently detects and removes DNA lesions within the large nuclear macromolecular complex called chromatin. The condensed nature of chromatin inhibits many DNA metabolizing activities, including NER. In order to promote efficient repair, detection of a lesion not only has to activate the NER pathway but also chromatin remodeling. In general, such remodeling is thought on the one hand to precede NER, thus allowing repair proteins to efficiently access DNA. On the other hand, after completion of the repair, the chromatin must be returned to its previous undamaged state. Chromatin remodeling can refer to three separate but interconnected processes, histone post-translational modifications, insertion of histone variants and histone displacement (including nucleosome sliding). Here we review current knowledge, and speculate about current unknowns, regarding those chromatin remodeling activities that physically displace histones before, during and after NER.
International Journal of Molecular Sciences 01/2012; 13(10):13322-37. · 2.60 Impact Factor
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Nature Structural & Molecular Biology 01/2012; 19(1):5-7. · 12.71 Impact Factor
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William E Pierceall,
Kam M Sprott,
Tuomas Heikkinen,
Paivi Heikkila,
Lakshmi Alaparthi,
Kristiina Aittomaki,
Mohammed Al-Adhami,
Vivian Villegas-Bergazzi,
Jane L Meyer,
Jeffery L Kutok,
Jirina Bartkova, Jiri Bartek,
Heli Nevanlinna,
David T Weaver,
Carl Blomqvist
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ABSTRACT: Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P ≤ .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue.
Human pathology 12/2011; 43(9):1363-75. · 3.03 Impact Factor
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ABSTRACT: Rab5 is a small GTPase known to regulate vesicular trafficking during interphase. Here, we show that Rab5 also plays an unexpected role during mitotic progression. RNAi-mediated silencing of Rab5 caused defects in chromosome congression and extensive prometaphase delay, and it correlated with a severe reduction in the localization of the centromere-associated protein CENP-F to kinetochores. CENP-F is a component of the nuclear matrix required for chromosome congression that, at mitotic entry, localizes to the nuclear envelope and assembles on kinetochores, contributing to the establishment of kinetochore microtubule interactions. We found that Rab5 forms a complex with a subset of CENP-F in mitotic cells and regulates the kinetics of release of CENP-F from the nuclear envelope and its accumulation on kinetochores. Simultaneous depletion of both Rab5 and CENP-F recapitulated the mitotic defects caused by silencing of either Rab5 or CENP-F alone, indicating epistatic roles for these two proteins in the pathway that orchestrates chromosome congression. These results reveal the involvement of Rab5 in the proper execution of mitotic programs whose deregulation can undermine chromosomal stability.
Proceedings of the National Academy of Sciences 10/2011; 108(42):17337-42. · 9.68 Impact Factor
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ABSTRACT: Following the discovery in 1998 of γ-H2AX, the first histone modification induced by DNA damage, interest in the changes to chromatin induced by DNA damage has exploded, and a vast amount of information has been generated. However, there has been a discrepancy between our rapidly advancing knowledge of how chromatin responds to DNA damage and the understanding of why cells mobilize large segments of chromatin to protect the genome against destabilizing effects posed by tiny DNA lesions. Recent research has provided insights into these issues and suggests that chromatin responses induced by DNA damage are not simply the accumulation of 'nuclear foci' but are mechanisms required to guard genome integrity.
Nature Cell Biology 10/2011; 13(10):1161-9. · 19.49 Impact Factor
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ABSTRACT: Although events associated with replication stress have long formed the cornerstone of checkpoint activation, questions remain about how cells maintain the integrity of replicating genomes. Now, Bermejo et al. (2011) identify a mechanism directly linking checkpoint function to the relief of topological tension at nuclear pore tethered genes.
Cell 07/2011; 146(2):189-91. · 32.40 Impact Factor
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Maral Jamshidi,
Jirina Bartkova,
Dario Greco,
Johanna Tommiska,
Rainer Fagerholm,
Kristiina Aittomäki,
Johanna Mattson,
Kenneth Villman,
Radek Vrtel,
Jiri Lukas,
Päivi Heikkilä,
Carl Blomqvist, Jiri Bartek,
Heli Nevanlinna
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ABSTRACT: NQO1 participates in cellular defense against oxidative stress and regulates apoptosis via p53- and NFκB-mediated pathways. We have previously found that homozygous missense variant NQO1*2 (rs1800566) predicts poor survival among breast cancer patients, particularly after anthracycline-based adjuvant chemotherapy. Here, we investigated NQO1 and NFκB protein expression and global gene expression profiles in breast tumors with correlation to tumor characteristics and survival after adjuvant chemotherapy. We used immunohistochemical analysis of tissue microarrays to study NQO1 and NFκB expression in two series of tumors: 1000 breast tumors unselected for treatment and 113 from a clinical trial comparing chemotherapy regimens after anthracycline treatment in advanced breast cancer. We used gene expression arrays to define genes co-expressed with NQO1 and NFκB. NQO1 and nuclear NFκB were expressed in 83% and 11% of breast tumors, and correlated inversely (P = 0.012). NQO1 protein expression was associated with estrogen receptor (ER) expression (P = 0.011), whereas 34.5% of NFκB-nuclear/activated tumors were ER negative (P = 0.001). NQO1 protein expression and NFκB activation showed only trends, but no statistical significance for patient survival or outcome after anthracycline treatment. Gene expression analysis highlighted 193 genes that significantly correlated with both NQO1 and NFκB in opposite directions, consistent with the expression patterns of the two proteins. Inverse correlation was found with genes related to oxidation/reduction, lipid biosynthesis and steroid metabolism, immune response, lymphocyte activation, Jak-STAT signaling and apoptosis. The inverse relationship between NQO1 protein expression and NFκB activation, underlined also by inverse patterns of association with ER and gene expression profiles of tumors, suggests that NQO1-NFκB interaction in breast cancer is different from several other tissue types, possibly due to estrogen receptor signaling in breast cancer. Neither NQO1 nor NFκB protein expression appear as significant prognostic or predictive markers in breast cancer.
Breast Cancer Research and Treatment 06/2011; 132(3):955-68. · 4.43 Impact Factor
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ABSTRACT: Centrosome abnormalities occur commonly in cancer, and contribute to chromosomal instability and tumorigenesis. New evidence on a phylogenetically conserved mechanism termed 'centrosomal clustering' provides exciting insights into how cells with supernumerary centrosomes adapt to avoid lethal multipolar divisions. Here, we highlight the emerging molecular basis of centrosome clustering, and its impact on asymmetric divisions of stem cells, chromosomal (in)stability and malignant transformation. Finally, pharmacological inhibition of centrosome clustering promises to selectively target tumor cells.
Molecular oncology 05/2011; 5(4):324-35. · 4.10 Impact Factor
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ABSTRACT: CHEK2 gene mutations occur in a subset of patients with familial breast cancer, acting as moderate/low penetrance cancer susceptibility alleles. Although CHEK2 is no longer recognized as a major determinant of the Li-Fraumeni syndrome, a hereditary condition predisposing to cancer at multiple sites, it cannot be ruled out that mutations of this gene play a role in malignancies arising in peculiar multi-cancer families. To assess the contribution of CHEK2 to the breast cancer/sarcoma phenotype, we screened for germ-line sequence variations of the gene among 12 probands from hereditary breast/ovarian cancer families with one case of sarcoma that tested wild-type for mutations in the BRCA1, BRCA2, and TP53 genes. Two cases harbored previously unreported mutations in CHEK2, the c.507delT and c.38A>G, leading to protein truncation (p.Phe169LeufsX2) and amino acid substitution (p.His13Arg), respectively. These mutations were not considered common polymorphic variants, as they were undetected in 230 healthy controls of the same ethnic origin. While the c.38A>G encodes a mutant protein that behaves in biochemical assays as the wild-type form, the c.507delT is a loss-of-function mutation. The identification of two previously unreported CHEK2 variants, including a truncating mutation leading to constitutional haploinsufficiency, in individuals belonging to families selected for breast cancer/sarcoma phenotype, supports the hypothesis that the CHEK2 gene may act as a factor contributing to individual tumor development in peculiar familial backgrounds.
Breast Cancer Research and Treatment 05/2011; 130(1):207-15. · 4.43 Impact Factor
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ABSTRACT: Centrosomes are central regulators of mitosis that are often amplified in cancer cells. Centrosomes function both as organizers of the mitotic spindle and as reaction centers to trigger activation of Cdk1 and G(2)/M transition in the cell cycle, but their functional organization remains incomplete. Recent proteomic studies have identified novel components of the human centrosome including Cep63, a protein of unknown function that Xenopus studies have implicated in mitotic spindle assembly and spindle inactivation after DNA damage. Here, we report that human Cep63 binds to and recruits Cdk1 to centrosomes, and thereby regulates mitotic entry. RNAi-mediated Cep63 depletion in U2OS cancer cells induced polyploidization through mitotic skipping. Elicitation of this phenotype was associated with downregulation of centrosomal Cdk1, mimicking the phenotype induced by direct depletion of Cdk1. In contrast, Cep63 overexpression induced de novo centrosome amplification during cell-cycle interphase. Induction of this phenotype was suppressible by cell treatment with the Cdk inhibitor roscovitine. In a survey of 244 neuroblastoma cases, Cep63 mRNA overexpression was associated with MYCN oncogene amplification and poor prognosis. In cultured cells, Cep63 overexpression was associated with an enhancement in replication-induced DNA breakage. Together, our findings define human Cep63 as a centrosomal recruitment factor for Cdk1 that is essential for mitotic entry, providing a physical link between the centrosome and the cell-cycle machinery.
Cancer Research 03/2011; 71(6):2129-39. · 7.86 Impact Factor
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ABSTRACT: Cellular senescence, an irreversible proliferation arrest evoked by stresses such as oncogene activation, telomere dysfunction, or diverse genotoxic insults, has been implicated in tumor suppression and aging. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), nuclear DNA domains stained densely by DAPI and enriched for histone modifications including lysine9-trimethylated histone H3. While cellular senescence occurs also in premalignant human lesions, it is unclear how universal is SAHF formation among various cell types, under diverse stresses, and whether SAHF occur in vivo. Here, we report that human primary fibroblasts (BJ and MRC-5) and primary keratinocytes undergoing replicative senescence, or premature senescence induced by oncogenic H-Ras, diverse chemotherapeutics and bacterial cytolethal distending toxin, show differential capacity to form SAHF. Whereas all tested cell types formed SAHF in response to activated H-Ras, only MRC-5, but not BJ fibroblasts or keratinocytes, formed SAHF under senescence induced by etoposide, doxorubicin, hydroxyurea, bacterial intoxication or telomere attrition. In addition, DAPI-defined SAHF were detected on paraffin sections of Ras-transformed cultured fibroblasts, but not human lesions at various stages of tumorigenesis. Overall, our results indicate that unlike the widely present DNA damage response marker γH2AX, SAHF is not a common feature of cellular senescence. Whereas SAHF formation is shared by diverse cultured cell types under oncogenic stress, SAHF are cell-type-restricted under genotoxin-induced and replicative senescence. Furthermore, while the DNA/DAPI-defined SAHF formation in cultured cells parallels enhanced expression of p16(ink4a) , such 'prototypic' SAHF are not observed in tissues, including premalignant lesions, irrespective of enhanced p16(ink4a) and other features of cellular senescence.
Cell cycle (Georgetown, Tex.) 02/2011; 10(3):457-68. · 5.36 Impact Factor
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Claudia Lukas,
Velibor Savic,
Simon Bekker-Jensen,
Carsten Doil,
Beate Neumann,
Ronni Sølvhøj Pedersen,
Merete Grøfte,
Kok Lung Chan,
Ian David Hickson, Jiri Bartek,
Jiri Lukas
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ABSTRACT: Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations.
Nature Cell Biology 02/2011; 13(3):243-53. · 19.49 Impact Factor
